Aspirin attenuates vinorelbine-induced endothelial inflammation via modulating SIRT1/AMPK axis.

Vinorelbine (VNR), a semisynthetic vinca alkaloid acquired from vinblastine, is frequently used as the candidate for intervention of solid tumors. Nevertheless, VNR-caused endothelial injuries may lead a mitigative effect of clinical treatment efficiency. A growing body of evidence reveals that aspirin is a potent antioxidant and anti-inflammation drug. We investigated whether aspirin attenuate VNR-induced endothelial dysfunction. Human endothelial cells (EA.hy 926) were treated with VNR to cause endothelial inflammation. Western blotting, ROS assay, ELISA were used to confirm the anti-inflammatory effect of aspirin. We confirmed that VNR suppresses SIRT1 expression, reduced LKB1 and AMPK phosphorylation as well as enriched PKC activation in treated endothelial cells. Furthermore, the membrane translocation assay displayed that the levels of NADPH oxidase subunits p47phox and Rac-1 in membrane fractions of endothelial cells were higher in cells that had been treated with VNR for than in untreated cells. We corroborated that treatment of Aspirin significantly diminishes VNR-repressed SIRT1, LKB1 and AMPK phosphorylation and VNR-promoted NADPH oxidase activation, however, those findings were vanished by SIRT1 and AMPK siRNAs. Our data also shown that Aspirin represses VNR-activated TGF-beta-activated kinase-1 (TAK1) activation, inhibited the interaction of TAK1/TAK-binding protein1 (TAB1), suppressed NF-kappa B activation and pro-inflammatory cytokine secretion. We demonstrated a novel connection between VNR-caused oxidative damages and endothelial dysfunction, and provide further insight into the protective effects of aspirin in VNR-caused endothelial dysfunction.

Combination treatment with fulvestrant and various cytotoxic agents (doxorubicin, paclitaxel, docetaxel, vinorelbine, and 5-fluorouracil) has a synergistic effect in estrogen receptor-positive breast cancer.

Patients with estrogen receptor (ER)-positive breast cancers have a better prognosis than those with ER-negative breast cancers, but often have low sensitivity to chemotherapy and a limited survival benefit. We have previously shown a combination effect of taxanes and fulvestrant and suggested that this treatment may be useful for ER-positive breast cancer. In this study, we evaluated the effects of combinations of hormone drugs and chemotherapeutic agents. In vitro, the effects of combinations of five chemotherapeutic agents (doxorubicin, paclitaxel, docetaxel, vinorelbine, and 5-fluorouracil) and three hormone drugs (fulvestrant, tamoxifen, and 4-hydroxytamoxifen) were examined in ER-positive breast cancer cell lines using CalcuSyn software. Changes in chemoresistant factors such as Bcl2, multidrug resistance-associated protein 1, and microtubule-associated protein tau were also examined after exposure of the cells to hormone drugs. In vivo, tumor sizes in mice were evaluated after treatment with docetaxel or doxorubicin alone, fulvestrant alone, and combinations of these agents. Combination treatment with fulvestrant and all five chemotherapeutic agents in vitro showed synergistic effects. In contrast, tamoxifen showed an antagonistic effect with all the chemotherapeutic agents. 4-Hydroxytamoxifen showed an antagonistic effect with doxorubicin and 5-fluorouracil, but a synergistic effect with taxanes and vinorelbine. Regarding chemoresistant factors, Bcl2 and microtubule-associated protein tau were downregulated by fulvestrant. In vivo, a combination of fulvestrant and docetaxel had a synergistic effect on tumor growth, but fulvestrant and doxorubicin did not show this effect. In conclusion, fulvestrant showed good compatibility with all the evaluated chemotherapeutic agents, and especially with docetaxel, in vitro and in vivo.

Resveratrol confers resistance against taxol via induction of cell cycle arrest in human cancer cell lines.

Resveratrol, which is highly concentrated in the skin of grapes and is abundant in red wine, has been demonstrated to account for several beneficial properties, including antioxidant, anticoagulant, anti-inflammatory and anticancer effects. Taxol is a microtubule-stabilizing drug that has been extensively used as effective chemotherapeutic agents in the treatment of solid tumors. Here, we investigated whether the combination of the two compounds would yield increased antitumor efficacy in human cancer cells. Unexpectedly, resveratrol effectively prevented tumor cell death induced by taxol in 5637 bladder cancer cells. This pronounced antagonistic function of resveratrol against taxol was associated with changes in multiple signal transduction pathways, but not with tubulin polymerization. Importantly, cell cycle analysis showed that resveratrol prevented the cells from entering into mitosis, the phase in which taxol exerts its action. Furthermore, resveratrol blocked the cytotoxic effects of vinblastine but not cisplatin in 5637 cells. Interestingly, resveratrol pre-treatment followed by taxol resulted in synergistic cytotoxicity. Finally, we extended our studies to various human cancer cell lines. Taken together, our results indicate that resveratrol may have the potential to negate the therapeutic efficacy of taxol and suggest that consumption of resveratrol-related products may be contraindicated during cancer therapy with taxol.

Microtubule binding and disruption and induction of premature senescence by disorazole C(1).

Disorazoles comprise a family of 29 macrocyclic polyketides isolated from the fermentation broth of the myxobacterium Sorangium cellulosum. The major fermentation product, disorazole A(1), was found previously to irreversibly bind to tubulin and to have potent cytotoxic activity against tumor cells, possibly because of its highly electrophilic epoxide moiety. To test this hypothesis, we synthesized the epoxide-free disorazole C(1) and found it retained potent antiproliferative activity against tumor cells, causing prominent G(2)/M phase arrest and inhibition of in vitro tubulin polymerization. Furthermore, disorazole C(1) produced disorganized microtubules at interphase, misaligned chromosomes during mitosis, apoptosis, and premature senescence in the surviving cell populations. Using a tubulin polymerization assay, we found disorazole C(1) inhibited purified bovine tubulin polymerization, with an IC(50) of 11.8 +/- 0.4 microM, and inhibited [3H]vinblastine binding noncompetitively, with a K(i) of 4.5 +/- 0.6 microM. We also found noncompetitive inhibition of [3H]dolastatin 10 binding by disorazole C(1), with a K(i) of 10.6 +/- 1.5 microM, indicating that disorazole C(1) bound tubulin uniquely among known antimitotic agents. Disorazole C(1) could be a valuable chemical probe for studying the process of mitotic spindle disruption and its relationship to premature senescence.

Stereoselective conversion of anhydrovinblastine into vinblastine utilizing an anti-vinblastine monoclonal antibody as a chiral mould.

Dimeric indole alkaloid, anhydrovinblastine, which can be obtained from catharanthine and vindoline in a high yield, was converted stereoselectively into vinblastine through alternating oxidation-reduction with oxygen and NaBH(3)CN in the presence of anti-vinblastine monoclonal antibody.

From bench to bedside: utilization of an in vitro model to predict potential drug-drug interactions in the kidney: the digoxin-mifepristone example.

Drug interactions are a common source of drug-induced toxicity. For drugs with narrow therapeutic windows, such as digoxin, an understanding of the potential mechanisms by which drugs might interact is essential to clinical practice. This article describes the utility of a renal tubular cell culture model in the prediction of drug interactions involving P-glycoprotein. Digoxin is a cardiac glycoside that undergoes active secretion in the renal tubules by the MDR1 (P-glycoprotein) drug efflux pump. Mifepristone (RU486) is a recently introduced abortifacient that is largely unstudied in terms of drug-drug interactions. The authors used an in vitro model to study the effects of mifepristone on the renal tubular secretion and cellular uptake of digoxin by Madin-Darby canine kidney (MDCK) cells. Mifepristone significantly inhibited the renal tubular secretion of digoxin (p = 0.0005), without interfering with its ability to enter the renal tubular cell. Similar results were found with the P-glycoprotein substrate vinblastine. The findings suggest that drug interactions may result if mifepristone is administered with P-glycoprotein substrates, highlighting the usefulness of this model in the study of not only common but also rare combinations of drugs.

Modulation of vinca-alkaloid induced P-glycoprotein expression by indole-3-carbinol.

The over-expression of mdr-1 gene transcript P-glycoprotein (P-gp), responsible for multiple drug resistance, is one of the major obstacles in cancer chemotherapy. In the present study, indole-3-carbinol (I3C), a well-known chemopreventive agent present in cruciferous vegetables, has been evaluated for its potential to modulate the over-expression of P-gp induced by vinblastine or vincristine, which are known inducers of mdr-1 gene. The results revealed that I3C significantly reversed the over-expression of P-gp in vinca-alkaloid induced drug resistance as evident by Western blotting using monoclonal antibody (clone JSB1). Quantization of immunostained tissue sections using image analysis technique revealed that vinblastine/ vincristine induced overexpression of P-gp was effectively reversed by I3C. The present investigation suggests that I3C can significantly inhibit the P-gp over-expression and may have utility as a dietary adjuvant in the treatment of cancer for the reversal of multiple drug resistance.

Vindoline biosynthesis is transcriptionally blocked in Catharanthus roseus cell suspension cultures.

Catharanthus roseus cell cultures were exposed to different conditions in order to induce alkaloid metabolism. The exposure to jasmonate and fungal elicitors resulted in the transcriptional activation of tryptophan decarboxylase and in the accumulation of the monoterpenoid indole alkaloids ajmalicine and catharanthine, but not of vindoline. The inability of the cell cultures to produce vindoline was related to a lack of expression of the desacetoxyvindoline 4-hydroxylase (D4h) gene. Southern blot analysis revealed that D4h gene was not lost in the cell cultures.

Characterization of the interaction of TZT-1027, a potent antitumor agent, with tubulin.

TZT-1027, a derivative of dolastatin 10 isolated from the Indian Ocean sea hare Dolabella auricularia in 1987 by Pettit et al., is a potent antimicrotubule agent. We have compared the activity of TZT-1027 with that of dolastatin 10 as well as the vinca alkaloids vinblastine (VLB), vincristine (VCR) and vindesine (VDS). TZT-1027 and dolastatin 10 inhibited microtubule polymerization concentration-dependently at 1 - 100 microM with IC50 values of 2.2 +/- 0.6 and 2.3 +/- 0.7 microM, respectively. VLB, VCR and VDS inhibited microtubule polymerization at 1 - 3 microM with IC50 values of 2.7 +/- 0.6, 1.6 +/- 0.4 and 1.6 +/- 0.2 microM, respectively, but showed a slight decrease in inhibitory effect at concentrations of 10 microM or more. TZT-1027 also inhibited monosodium glutamate-induced tubulin polymerization concentration-dependently at 0.3 - 10 microM, with an IC50 of 1.2 microM, whereas VLB was only effective at 0.3 - 3 microM, with an IC50 of 0.6 microM, and caused so-called "aggregation" of tubulin at 10 microM. Scatchard analysis of the binding data for [(3)H]VLB suggested one binding site (Kd 0.2 +/- 0.04 microM and Bmax 6.0 +/- 0.26 nM / mg protein), while that for [(3)H]TZT-1027 suggested two binding sites, one of high affinity (Kd 0.2 +/- 0.01 microM and Bmax 1.7 +/- 0.012 nM / mg protein) and the other of low affinity (Kd 10. 3 +/- 1.46 microM and Bmax 11.6 +/- 0.83 nM / mg protein). [(3)H]TZT-1027 was completely displaced by dolastatin 10 but only incompletely by VLB. [(3)H]VLB was completely displaced by dolastatin 10 and TZT-1027. Furthermore, TZT-1027 prevented [(3)H]VLB from binding to tubulin in a non-competitive manner according to Lineweaver-Burk analysis. TZT-1027 concentration-dependently inhibited both [(3)H]guanosine 5'-triphosphate (GTP) binding to and GTP hydrolysis on tubulin. VLB inhibited the hydrolysis of GTP on tubulin concentration-dependently to a lesser extent than TZT-1027, but no inhibitory effect of VLB on [(3)H]GTP binding to tubulin was evident even at 100 microM. Thus, TZT-1027 affected the binding of VLB to tubulin, but its binding site was not completely identical to that of VLB. TZT-1027 had a potent inhibitory effect on tubulin polymerization and differed from vinca alkaloids in its mode of action against tubulin polymerization.

'Accidental' anti-angiogenic drugs. anti-oncogene directed signal transduction inhibitors and conventional chemotherapeutic agents as examples.

A number of drugs currently being tested in clinical trials as possible angiogenesis inhibitors were not originally developed with the intention of suppressing tumour angiogenesis. Thalidomide and interferon alpha are obvious examples of such drugs. This list of 'accidental' angiogenesis inhibitors may include established agents such as conventional cytotoxic chemotherapeutic drugs as well as the new generation of anticancer drugs known as anti-oncoprotein signal transduction inhibitors. With respect to the former, the potential of such drugs to inhibit angiogenesis could be the result of their ability to cause collateral damaging effects on cycling endothelial cells found in newly formed blood vessels, or inhibiting other vital endothelial cell functions necessary for angiogenesis. The antitumour vascular side-effects of chemotherapy may be optimised by administering such drugs continuously on a more frequent (e.g. weekly or even daily) basis at levels well below the maximum tolerated dose (MTD), especially when this is done in combination with newly developed anti-angiogenic drugs such as vascular endothelial cell growth factor (VEGF) receptor blocking antibodies. This strategy may minimise or delay the problems of host toxicity and acquired drug resistance. The possibility of anti-angiogenic effects mediated by signal transduction inhibitors such as ras farnesyltransferase inhibitors (ras FTI's), or drugs which block receptor tyrosine kinases (e.g. ErbB2/neu) such as Herceptin, may be the consequence of such oncogenes inducing or upregulating various pro-angiogenic molecules such as VEGF (vascular endothelial cell growth factor) in tumour cells. Hence, treatment of tumour cells with such drugs can lead to downregulation of tumour cell-associated VEGF expression and this can contribute to an anti-angiogenic effect of the drug in vivo. In addition, some of these drugs may also affect certain 'activated' endothelial cell functions directly so as to block angiogenesis. An awareness of the potential of such conventional or experimental anticancer drugs to affect tumour growth through blockade or suppression of angiogenesis has implications for how anticancer drugs may be used clinically, either alone, or in combination with other drugs to optimally treat cancer.

Interactions of the sponge-derived antimitotic tripeptide hemiasterlin with tubulin: comparison with dolastatin 10 and cryptophycin 1.

The sponge-derived antimitotic tripeptide hemiasterlin was previously shown to inhibit tubulin polymerization. We have now demonstrated that hemiasterlin resembles most other antimitotic peptides in noncompetitively inhibiting the binding of vinblastine to tubulin (apparent K(i) value, 7.0 microM), competitively inhibiting the binding of dolastatin 10 to tubulin (apparent K(i) value, 2.0 microM), stabilizing the colchicine binding activity of tubulin, inhibiting nucleotide exchange on beta-tubulin, and inducing the formation of tubulin oligomers that are stable to gel filtration in the absence of free drug, even at low drug concentrations. The tubulin oligomerization reaction induced by hemiasterlin was compared to the reactions induced by dolastatin 10 and cryptophycin 1. Like dolastatin 10, hemiasterlin induced formation of a tubulin aggregate that had the morphological appearance primarily of ring-like structures with a diameter of about 40 nm, while the morphology of the cryptophycin 1 aggregate consisted primarily of smaller rings (diameter about 30 nm). However, the hemiasterlin aggregate differed from the dolastatin 10 aggregate in that its formation was not associated with turbidity development, and the morphology of the hemiasterlin aggregate (as opposed to the dolastatin 10 aggregate) did not change greatly when microtubule-associated proteins were present (tight coils and pinwheels are observed with dolastatin 10 but not with hemiasterlin or cryptophycin 1). Opacification of tubulin-dolastatin 10 mixtures was inhibited by hemiasterlin at 22 degrees C and stimulated at 0 degrees C, while cryptophycin 1 was inhibitory at both reaction temperatures.

Tubercidin stabilizes microtubules against vinblastine-induced depolymerization, a taxol-like effect.

A sensitive assay for the detection of microtubule-stabilizing agents [1] was used to screen an extensive collection of cyanobacterial and microalgal extracts. The hydrophilic extract of the cyanobacterium, Plectonema radiosum (UH isolate IC-70-1), exhibited microtubule-stabilizing activity. Bioassay-directed purification of the active compound yielded tubercidin (7-deazaadenosine), a potent cytotoxic nucleoside analog. Further studies revealed that tubercidin protected a population of cellular microtubules against vinblastine-induced depolymerization, a microtubule-stabilizing, taxol-like effect. The microtubule-stabilizing effect of tubercidin is dose dependent and limited by the cytotoxicity of the agent. Tubercidin represents another natural product that interacts with microtubules and is one of the few to cause microtubule stabilization.

The spongistatins, potently cytotoxic inhibitors of tubulin polymerization, bind in a distinct region of the vinca domain.

The highly cytotoxic, sponge-derived, antimitotic macrolide polyether spongistatin 1 has been previously shown to inhibit microtubule assembly, the binding of vinblastine and GTP to tubulin, and displacement of GDP bound in the exchangeable site of tubulin. We have now examined in detail inhibition by spongistatin 1 of both [3H]vinblastine and [3H]dolastatin 10 binding to tubulin. We found spongistatin 1 to be a noncompetitive inhibitor of the binding of both radiolabeled drugs to tubulin, in contrast to competitive patterns obtained with vincristine versus [3H]vinblastine and with a chiral isomer of dolastatin 10 versus [3H]dolastatin 10. Since dolastatin 10 is itself a noncompetitive inhibitor of vinca alkaloid binding to tubulin, this implies at least three distinct binding sites for the structurally complex and diverse natural products that interfere with each others binding to tubulin and with nucleotide exchange. Spongistatin 1, in contrast to both vinca alkaloids and peptide antimitotic agents like dolastatin 10, does not induce formation of a GTP-independent, morphologically distinctive polymer ("aggregate"). We also examined eight compounds closely related structurally to spongistatin 1 (spongistatins 2-9). The most distinctive in their properties were spongistatins 6 and 8. These two compounds, despite activity comparable to spongistatin 1 as inhibitors of tubulin polymerization and [3H]vinblastine binding, had much reduced activity as inhibitors of nucleotide exchange and [3H]dolastatin 10 binding. Spongistatins 1 and 6 were compared for effects on dolastatin 10-induced aggregate formation in conjunction with effects on [3H]dolastatin 10 binding. Spongistatin 6 was about 4-fold less active than spongistatin 1 as an inhibitor of aggregation and over 20-fold less active as an inhibitor of dolastatin 10 binding.

Symptom relief with MVP (mitomycin C, vinblastine and cisplatin) chemotherapy in advanced non-small-cell lung cancer.

The role of chemotherapy in the palliation of patients with advanced stage (IIIB and IV non-small-cell lung cancer (NSCLC) remains controversial. We have carried out a chemotherapy study emphasising symptom relief, a topic not normally discussed in previous similar studies. A total of 120 patients with locally advanced or metastatic non-small-cell lung cancer (NSCLC) were treated with a moderate-dose palliative chemotherapy regimen consisting of mitomycin C 8 mg m-2 i.v. on day 1 (alternate courses), vinblastine 6 mg m-2 i.v. on day 1 and cisplatin 50 mg m-2 i.v. on day 1 (MVP), repeating every 21 days for a maximum of six courses. Thirty-eight of 118 assessable patients (32%) achieved an objective response. Patients with locally advanced disease (stage IIIB) had a significantly better response rate (52%) than those with metastatic disease (25%) (P < 0.01). In 76 out of 110 (69%) patients, with tumour-related symptoms including 24 out of 31 patients (78%) with locally advanced disease, symptoms completely disappeared or substantially improved. In only 15 patients (14%) did symptoms progress during treatment. Symptomatic improvement was achieved after one course of chemotherapy in 61% and after two courses in 96% of responding patients. The schedule was well tolerated. Only 19% developed WHO grade 3/4 nausea/vomiting, and only 3% developed significant alopecia. Other toxicities were minimal. MVP is a pragmatic inexpensive chemotherapy regimen that offers useful symptom palliation in patients with advanced NSCLC and merits a 1-2 course therapeutic trial in such patients. The schedule should also be assessed as primary (neoadjuvant) chemotherapy before radical radiotherapy for locally advanced NSCLC in a randomised trial.

A sensitive assay for taxol and other microtubule-stabilizing agent.

The ability of taxol to protect microtubules in cultured human ovarian carcinoma cells from drug- and cold-induced depolymerization was characterized as a functional assay for microtubule stabilizing agents. Treatment of the cells with concentrations of vinblastine or colchicine of 50 nM or greater, or incubation at 4 degrees C resulted in complete depolymerization of cytoplasmic microtubules. Pretreatment with taxol for 3 h enabled the cells to maintain substantial numbers of microtubules following the application of vinblastine or colchicine. This protective effect was easily observed at 50 nM taxol, whereas taxol-induced microtubule bundling was observed only at concentrations of 500 nM or greater. Concentrations of taxol as low as 10 nM stabilized microtubules against cold-induced depolymerization. Therefore, protection of microtubules from drug- and cold-induced depolymerization provides a sensitive functional assay for taxol. These systems should be similarly effective in identifying novel compounds which stabilize microtubules.

Inhibition of P-glycoprotein-mediated vinblastine transport across HCT-8 intestinal carcinoma monolayers by verapamil, cyclosporine A and SDZ PSC 833 in dependence on extracellular pH.

The ability of the multidrug resistance modifiers R- and R,S-verapamil (VPL), cyclosporine A (CsA) and its non-immunosuppressive derivative SDZ PSC 833 (PSC 833) to inhibit P-glycoprotein (P-gp)-mediated transepithelial flux of tritiated vinblastine was investigated using tight and highly resistant (R > 1,400 omega cm2) monolayer cultures of intestinal adenocarcinoma-derived HCT-8 cells grown on permeable tissue-culture inserts. Apical addition of these chemosensitizers inhibited drug flux (137 pmol h-1 cm-2; range, 133-142 pmol h-1 cm-2) in the basal to apical secretory direction at clinically relevant concentrations, with PSC 833 showing the highest activity, exhibiting inhibition at concentrations as low as 10 ng/ml (9 nM). Acidification of the modulator-containing apical compartment to an extracellular pH (pHo) of 6.8 had no influence on MDR reversal by CsA at 1 microgram/ml (0.9 microM; flux inhibition, 52%) or by PSC 833 at 100 ng/ml (0.09 microM; flux inhibition, 60%), in contrast to R,S- and R-VPL, which showed decreased inhibition and caused less accumulation of vinblastine in HCT-8 cells under this condition (flux inhibition of 35% and 23%, respectively, at pHo 6.8 vs 50% and 43%, respectively, at pHo 7.5). P-gp-mediated rhodamine 123 efflux from dye-loaded single-cell suspensions of HCT-8 cells as measured by flow cytometry was not impeded at pHo 6.8 in comparison with pHo 7.5 in standard medium, but at low pHo the inhibitory activity of R-VPL (29% vs 60% rhodamine 123 efflux inhibition) was diminished significantly, again without a reduction in the effect of PSC 833 (rhodamine 123 flux inhibition, 75%). In conclusion, drug extrusion across polarised monolayers, which offer a relevant model for normal epithelia and tumour border areas, is inhibited by the apical presence of R,S- and R-VPL, CsA and PSC 833 at similar concentrations described for single-cell suspensions, resulting in increased (2.2- to 3.7-fold) intracellular drug accumulation. Functional apical P-gp expression, the absence of paracellular leakage and modulator-sensitive rhodamine 123 efflux in single HCT-8 cells indicate a P-gp-mediated transcellular efflux in HCT-8 monolayers. In addition to its high MDR-reversing capacity, the inhibitory activity of PSC 833 is not affected by acidic extracellular conditions, which reduce the VPL-induced drug retention significantly. As far as MDR contributes to the overall cellular drug resistance of solid tumours with hypoxic and acidic microenvironments, PSC 833 holds the greatest promise for clinical reversal of unresponsiveness to the respective group of chemotherapeutics.

Transepithelial secretion, cellular accumulation and cytotoxicity of vinblastine in defined MDCK cell strains.

Transepithelial vinblastine secretion in two defined MDCK strains displays saturation kinetics; (Strain 1) Km = 2.8 +/- 0.6 microM (six experiments), Vmax 35.9 +/- 1.93 pmol/cm2 per h (six experiments), Strain 2 Km 0.78 +/- 0.36 microM (three experiments), Vmax 12.1 +/- 4.5 pmol/cm2 per h (three experiments). Concentrations of vinblastine > 1 microM are associated with an increased passive vinblastine permeability (PA-B). This correlates with an increased transepithelial conductance/decreased permselectivity, suggesting that this may in part result from increased paracellular conductance. Verapamil inhibits vinblastine secretion, half-maximal inhibition of basal-to-apical flux (JB-A) is observed at 3.4 +/- 0.3 and 1.7 +/- 0.05 microM verapamil for Strain-1 and Strain-2 epithelial layers, respectively. Cellular accumulation of vinblastine across the apical membrane is small with respect to that across the basolateral surfaces. This polarity is unaffected by verapamil. The apical membranes, therefore, possess a low intrinsic permeability to vinblastine. Inhibition of cell growth by vinblastine is enhanced by verapamil. Both the effect of vinblastine, and its enhancement by verapamil, upon cell growth are reduced as initial cell seeding density increases.

Apoptosis induced by microtubule disrupting drugs in cultured human lymphoma cells. Inhibitory effects of phorbol ester and zinc sulphate.

The effects of the microtubule disrupting drugs (MDD) vinblastine, vincristine and colchicine on a human lymphoma cell line, BM 13674, were investigated. Twelve hours after administration of vinblastine (10(-3) mg/ml), vincristine (10(-2) mg/ml) or colchicine (10(-2) mg/ml), cell death with the characteristic morphology of apoptosis was observed in 71.6%, 82.2% and 76.9% of the cells respectively. The mode of death was confirmed as apoptotic by the occurrence of internucleosomal DNA cleavage, which was demonstrated by agarose gel electrophoresis. For the purpose of casting light on the mechanism involved, inhibition tests were performed on apoptosis induced by one of these drugs, vinblastine, using a phorbol ester (PDBu), zinc sulphate and cycloheximide. PDBu, an activator of protein kinase C, and zinc sulphate, a putative inhibitor of the endonuclease were thought to be responsible for internucleosomal DNA cleavage; both markedly reduced the induction of apoptosis. The protein synthesis inhibitor cycloheximide, on the other hand, had no inhibitory effect. Moreover, cycloheximide treatment per se enhanced apoptosis. This suggests that new protein synthesis is not required for the execution of vinblastine-induced apoptosis. Such a finding is in accord with recent reports suggesting that the "death program" within many cell types may be primed but unable to proceed due to concomitant production of specific "apoptotic inhibitors". It is suggested that phorbol esters prevent vinblastine-induced apoptosis in the BM 13674 cells by activating one or more of these specific "apoptotic inhibitors", possibly by means of PKC-mediated phosphorylation.

Stereoisomers of calcium antagonists which differ markedly in their potencies as calcium blockers are equally effective in modulating drug transport by P-glycoprotein.

The (-)-isomer of verapamil is 10-fold more potent as a calcium antagonist than the (+)-isomer. However, both enantiomers are equally effective in increasing cellular accumulation of anticancer drugs [Gruber et al., Int J Cancer 41: 224-226, 1988]. In addition to verapamil, there exists a wide variety of stereoisomers with phenylalkylamines and dihydropyridine structures which markedly differ in their potency as calcium antagonists. We have tested these drugs for their ability to increase intracellular accumulation of [3H]vinblastine ([3H]VBL) in a doxorubicin-resistant cell line (F4-6RADR) derived from the Friend mouse leukemia cell line (F4-6P) and in COS-7 monkey kidney cells. Both cell types express substantial amounts of multidrug resistance gene 1 mRNA and P-glycoprotein as revealed by RNA and immuno blot analysis. The enantiomers with phenylalkylamine structures [(+/-)-verapamil; (+/-)-devapamil; (+/-)-emopamil)] and with dihydropyridine structures [(+/-)-isradipine; (+/-)-nimodipine; (+/-)-felodipine; (+/-)-nitrendipine; (+/-)-niguldipine] increased [3H]VBL accumulation in both cell lines at micromolar concentrations. Although the stereoisomers of these drugs differ markedly in their potency as calcium channel blockers they were about equally effective in increasing VBL levels in the cells. There was no substantial difference in the potencies of the phenylalkylamine drugs in affecting cellular [3H]VBL transport. Major potency differences, however, were observed in the dihydropyridine drug series with the niguldipine isomers as the most effective drugs. Moreover, the niguldipine enantiomers were equally as effective in reversing VBL resistance in F4-6RADR cells as were the verapamil enantiomers. Since (-)-niguldipine (B859-35) displays a 45-fold lower affinity for calcium channel binding sites than (+)-niguldipine, but is equally potent in inhibiting drug transport by P-glycoprotein and in reversing drug resistance, it may be, in addition to (+)-verapamil, another useful candidate drug for the treatment of multidrug resistance in cancer patients.

New potent verapamil derivatives that reverse multidrug resistance in human renal carcinoma cells and in transgenic mice expressing the human MDR1 gene.

Multidrug resistance in human renal cell carcinoma is mainly caused by expression of the MDR1 gene and is characterized by a broad spectrum cross resistance to many natural product chemotherapeutic agents. This resistance can be overcome by applying chemosensitizers which inhibit the function of the MDR1 gene product P-glycoprotein. The development of new reversing agents with fewer side effects and a higher potency in modifying resistance is a high priority of research on drug resistance. We have evaluated four new verapamil derivatives on 21 primary human renal cell carcinomas in vitro, and also tested them in an MDR-transgenic mice model. These mice express the human MDR1 gene in their bone marrow cells and measurement of their white blood counts provides a simple, rapid and reliable system to screen for the potency of MDR-reversing agents in vivo. We demonstrate here that all four drugs are effective in reversing multidrug resistance in primary cultures of human renal cell carcinomas when used in combination with vinblastine chemotherapy, and to a lesser extent with doxorubicin or daunomycin chemotherapy. Our in vivo data indicate that two of these reversing agents display low toxicity at high concentrations and are more effective at low, clinically achievable concentrations, than the other two drugs and R-verapamil. These results make the two new drugs attractive candidates to be taken into clinical trials.